Cystic fibrosis (CF) is a human genetic disease of epithelia. Although the survival rate of those suffering with cystic fibrosis has improved in recent years, the median age for patient survival is still only about 25-30 years despite intensive supportive and prophylactic treatment. Today cystic fibrosis remains the most common congenital disease among Caucasians, where it has a prevalence of about 1 in 2,000 live births and is uniformly fatal. Nearly all patients suffering from the disease develop chronic progressive disease of the respiratory system, the most common cause of death being pulmonary disease. In the majority of cases, pancreatic dysfunction occurs; hepatobiliary and genitourinary disease are also frequent. Because of the multi-system clinical manifestations of the disease, current methods of treatment for the disease have focused on therapeutic approaches to reduce the symptoms of cystic fibrosis.
It is now known that the disease is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation-regulated Clxe2x88x92 channel located in the apical membrane of involved epithelia. Also, much has been discovered about how CF-associated mutations disrupt protein function, thereby disrupting Clxe2x88x92 transport across CF epithelia.
Despite any advances, the pathogenesis of CF lung disease is still not understood. Lung disease is characterized by bacterial colonization and chronic airway infection. Many organisms can be involved, but Pseudomonas aeruginosa and Staphylococcus aureus are particularly prominent. Chronic bacterial infections progressively destroy the lung, and may ultimately lead to respiratory failure.
Airway infections currently cause most of the morbidity and mortality in cystic fibrosis (CF) (Taussig, L. M. 1984. Cystic Fibrosis. Georg Thieme Verlag Stuttgart, N.Y.; Davis, P. B. 1993. Pathophysiology of the Lung Disease in Cystic Fibrosis. In Cystic Fibrosis. P. B. Davis, editor. Marcel Dekker, Inc., New York. 193-218; Welsh, M. J., L. -C. Tsui, T. F. Boat and A. L. Beaudet. 1995. Cystic Fibrosis. In The Metabolic and Molecular Basis of Inherited Disease. C. R. Scriver, A. L. Beaudet, W. S. Sly and D. Valle, editors. McGraw-Hill, Inc., New York. 3799-3876; Burns, J. L., B. W. Ramsey, and A. L. Smith. 1993. Clinical manifestations and treatment of pulmonary infections in cystic fibrosis. Adv. Pediatr. Infect. Dis. 8:53-56). Infections begin early in the course of disease, are nearly impossible to eradicate, and together with the resulting exuberant inflammation destroy the lung. The pathogenesis of CF airway infection involves a host defense defect that is restricted to the airways; other organs are not infected, and when non-CF lungs are transplanted into a CF patient, they do not become infected (Taussig, L. M. 1984. Cystic Fibrosis. Georg Thieme Verlag Stuttgart, New York; Davis, P. B. 1993. Pathophysiology of the Lung Disease in Cystic Fibrosis. In Cystic Fibrosis. P. B. Davis, editor. Marcel Dekker, Inc., New York. 193-218; Welsh, M. J., L. C. Tsui, T. F. Boat, and A. L. Beaudet. 1995. Cystic Fibrosis. In The Metabolic and Molecular Basis of Inherited Disease. C. R. Scriver, A. L. Beaudet, W. S. Sly and D. Valle, editors. McGraw-Hill, Inc., New York. 3799-3876; Davis, P. B., M. Drumm and M. W. Konstan. 1996, Cystic Fibrosis. Am. J. Respir. Crit. Care Med. 154:1229-1256; Wine, J. J. 1999. The genesis of cystic fibrosis lung disease. J. Clin. Invest. 103:309-312; Pilewski, J. M., and R. A. Frizzel. 1999. Role of CFTR in Airway Disease. Physiol Rev. 79:S215-S255; Quinton, P. 1999. Physiological basis of cystic fibrosis: a historical perspective. Physiol. Rev. 79:S3-S22; Accurso, F. J. 1997. Early pulmonary disease in cystic fibrosis. Curr. Opin. Pulm. Med. 3:400-403). Early in the disease, many different organisms infect the airways, but with time Staphylococcus aureus and Pseudomonas aeruginosa predominate (Burns, J. L., J. Emerson, J. R. Stapp, D. L. Yim, J. Krzewinski, L. Louden, B. W. Ramsey and C. R. Clausen. 1998. Microbiology of sputum from patients at cystic fibrosis centers in the United States. Clin. Infect. Dis. 27:158-163).
The pathogenesis of CF airway infections and link mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) Clxe2x88x92 channel to the propensity for infection may be explained as follows (Smith, J. J., S. M. Travis, E. P. Greenberg and M. J. Welsh. 1996. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell. 85:229-236; and erratum 287(222)). The thin layer of airway surface liquid (ASL) contains many antimicrobial substances including lysozyme, lactoferrin, secretory leukoproteinase inhibitor (SLPI), human beta defensins 1 and 2, secretory phospholipase A2, and the cathelicidin LL-37 (Arnold, R. R., M. Brewer and J. J. Gauthier. 1980. Bactericidal activity of human lactoferrin: Sensitivity of a variety of microorganisms. Infect. Immun. 28:893-898; Jacquot, J., J. M. Tournier, T. G. Carmona, E. Puchelle, J. P. Chazalette and P. Sadoul. 1983. Proteins of bronchial secretions in mucoviscidosis. Role of infection. Bull. Eur. Physiopathol. Respir. 19:453-458; Thompson, A. B., T. Bohling, F. Payvandi, and S. I. Rennard. 1990. Lower respiratory tract lactoferrin and lysozyme arise primarily in the airways and are elevated in association with chronic bronchitis. J. Lab. Clin. Med. 115:148-158; Hiemstra, P. S., R. J. Maassen, J. Stolk, R. Heinzel-Wieland, G. J. Steffens, and J. H. Dijkman. 1996. Antibacterial activity of antileukoprotease. Infect. Immun. 64:4520-4524; Zhao, C., I. Wang, and R. I. Lehrer. 1996. Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett. 396:319-322; McCray, P. B. and L. Bentley. 1997. Human airway epithelia express a xcex2-defensin. Am. J. Respir. Cell. Mol. Biol. 16:343-349; Goldman, M. J., G. M. Anderson, E. D. Stolzenberg, U. P. Kari, M. Zasloff, and J. M. Wilson. 1997. Human xcex2-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell. 88:553-560; Bals, R., X. Wang, Z. Wu, T. Freeman, V. Bafna, M. Zasloff and J. M. Wilson. 1998. Human xcex2-defensin-2 is a salt-sensitive peptide antibiotic expressed in human lung. J Clin Invest. 102; Diamond, G., and C. L. Bevins. 1998. Beta-defensins: endogenous antibiotics of the innate host defense response. Clin. Immunol. Immunopath. 88:221-225; Bals, R., X. Wang, M. Zasloff and J. M. Wilson. 1998. The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc. Nat""l. Acad. Sci. USA. 95:9541-9546; Travis, S. M., B. A. D. Conway, J. Zabner, J. J. Smith, N. N. Anderson, P. K. Singh, E. P. Greenberg, and M. J. Welsh. 1999. Activity of Abundant Antimicrobials of the Human Airway. Am. J. Respir. Cell Mol. Biol. 20:872-879; Singh, P. K., H. P. Jia, K. Wiles, J. Hesselberth, L. Liu, B. D. Conway, E. Valore, M. J. Welsh, T. Ganz, B. F. Tack and P. B. J. McCray. 1998. Constitutive and inducible expression of xcex2-defensin antimicrobial peptides by human airway epithelia. Unpublished.). These agents acting alone and synergistically form part of the local pulmonary host defense system, killing the small numbers of bacteria that are constantly being deposited on the airway surface. Importantly, an increase in salt concentration inhibits the antibacterial activity of nearly all these agents and attenuates synergy between agents (Goldman, M. J., G. M. Anderson, E. D. Stolzenberg, U. P. Kari, M. Zasloff, and J. M. Wilson. 1997. Human xcex2-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell. 88:553-560; Bals, R., X. Wang, Z.Wu, T. Freeman, V. Bafna, M. Zasloff and J. M. Wilson. 1998. Human xcex2-defensin-2 is a salt-sensitive peptide antibiotic expressed in human lung. J. Clin. Invest. 102; Travis, S. M., B. A. D. Conway, J. Zabner, J. J. Smith, N. N. Anderson, P. K. Singh, E. P. Greenberg, and M. J. Welsh. 1999. Activity of Abundant Antimicrobials of the Human Airway. Am. J. Respir. Cell Mol. Biol. 20:872-879; Singh, P. K., H. P. Jia, K. Wiles, J. Hesselberth, L. Liu, B. D. Conway, E. Valore, M. J. Welsh, T. Ganz, B. F. Tack, and P. B. J. McCray. 1998. Constitutive and inducible expression of xcex2-defensin antimicrobial peptides by human airway epithelia. Unpublished; Davies, R. C., A. Neuberger. and B. M. Wilson. 1969. The dependence of lysozyme on pH and ionic strength. Biochim. Biophys. Acta. 178:294-305; Millar, M. 1987. The susceptibility to lysozyme of b-lactamase-producing and non-producing derivatives of Staphylococcus aureus strain 1030. J. Med. Microbiol. 23:127-132; Valore, E. V., C. H. Park, A. J. Quayle, K. R. Wiles, P. B. J. McCray, and T. Ganz. 1998. Human xcex2-defensin-1: An antimicrobial peptide of urogenital tissues. J. Clin. Invest. 101:1633-1642; Singh, P., and M. J. Welsh. 1999. Components of airway surface fluid have synergistic antimicrobial activity. Pediatr. Pulmonol. Suppl 14:323). In CF, the loss of the CFTR Clxe2x88x92 channel leads to a higher ASL salt concentration which reduces antimicrobial potency, thereby impairing the innate immune system and predisposing to infection.
Evidence for an elevated salt concentration in CF ASL came from in vivo studies that collected tiny volumes ( less than 1 xcexcl) of ASL from trachea and bronchus of anesthetized subjects (Joris, L., I. Dab, and P. M. Quinton. 1993. Elemental composition of human airway surface fluid in healthy and diseased airways. Am. Rev. Respir. Dis. 148:1633-1637). Using an in vitro model of differentiated human airway epithelia and a non-invasive isotope method, at equilibrium, non-CF ASL was found to have Na+ and Clxe2x88x92 concentrations of xcx9c40-50 mM, whereas CF values were xcx9c85-95 mM (Zabner, J., J. J. Smith, P. H. Karp, J. H. Widdicombe, and M. J. Welsh. 1998. Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol. Cell. 2:397-403). In contrast, in vivo studies using filter paper to collect xcx9c20 xcexcl of liquid from bronchus (Knowles, M. R., J. M. Robinson, R. E. Wood, C. A. Pue, W. M. Mentz, G. C. Wager, J. T. Gatzy, and R. C. Boucher. 1997. Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J. Clin. Invest. 100:2588-2595; Hull, J., W. Skinner, C. Robertson, and P. Phelan. 1998. Elemental content of airway surface liquid from infants with cystic fibrosis. Am. J. Respir. Crit. Care Med. 157:10-14) led to the conclusion that ASL had NaCl concentrations equal to that of serum, and that there was no difference between CF and non-CF. A potential explanation for the difference between the studies is that the filter paper sampling technique may have altered ASL composition. Earlier studies showed that filter paper draws liquid from the serum; thus the more liquid collected, the more the contamination with serum (Erjefxc3xa4lt, I., and C. G. A. Persson. 1990. On the use of absorbing discs to sample mucosal surface liquids. Clin. Exp. All. 20:193-197). Additional studies done at equilibrium with in vitro models of human airway epithelia grown at the air-liquid interface (Widdicombe, J. H., H. Fischer, C. Y. -C. Lee, S. N. Uyekubo, and S. S. Miller. 1997. Elemental composition of airway surface liquid. Pediatric Pulmonology. Suppl. 14:74; Jacquot, J., O. Tabary, S. Baconnais, G. Balossier, D. Hubert, J. Couetil, and E. Puchelle. 1998. Highly increased levels of constitutive sodium chloride and C-X-C chemokines production by CF human bronchial submucosal gland cells. Pediatric Pulmonology. Suppl. 17:387) and xenografts of human airway epithelia (Goldman, M. J., G. M. Anderson, E. D. Stolzenberg, U. P. Kari, M. Zasloff, and J. M. Wilson. 1997. Human xcex2-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell. 88:553-560; Zhang, Y., and J. F. Engelhardt. 1999. Airway surface fluid volume and Cl content in cystic fibrosis and normal bronchial xenografts. Am. J. Physiol. 276:469-476; Baconnais, S., R. Tirouvanziam, J. M. Zahm, S. de Bentzmann, B. Peault, G. Balossier, and E. Puchelle. 1999. Ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts. Am. J. Respir. Cell. Mol. Biol. 20:605-611) indicate that the non-CF ASL NaCl concentration is much lower than that of serum, and that ASL salt concentrations are higher in CF. Consistent with this data, in vitro and in vivo studies in rodents indicate that the ASL NaCl concentration is much lower than that of serum (Cowley, E. A., K. Govindaraju, D. K. Lloyd, and D. H. Eidelman. 1997. Is mouse airway surface fluid hypotonic. Pediatric Pulmonology. Suppl. 14:233; Cowley. E. A., K. Govindaraju, D. K. Lloyd, and D. H. Eidelman. 1997. Airway surface fluid composition in the rat determined by capillary electrophoresis. American Physiological Society. 273:L895-L899; Bacconais, S., J. Zahm, L. Kilian, P. Bonhomme, D. Gobillard, A. Perchet, E. Puchelle, and G. Balossier. 1998. X-ray microanalysis of native airway surface liquid collected by cryotechnique. J. Microsc. 191:311-319; McCray, P. B. J., J. Zabner, H. P. Jia, M. J. Welsh, and P. S. Thorne. 1999. Efficient killing of inhaled bacteria in xcex94F508 mice: role of airway surface composition. Am. J. Physiol. 277:L183-L190).
Based on the salt sensitivity of endogenous antimicrobials and the elevated salt content in CF ASL, it was studied whether lowering the ASL NaCl concentration could help prevent CF airway infections. Several factors were considered. First, the airway epithelium is water permeable (Folkesson, H. G., M. A. Matthay, A. Frigeri and A. S. Verkman. 1996. Transepithelial water permeability in microperfused distal airways. J. Clin. Invest. 97:664-671). Consistent with this, when large volumes of liquid are placed on the apical surface, liquid absorption is isotonic (Zabner, J., J. J. Smith, P. H. Karp, J. H. Widdicombe, and M. J. Welsh. 1998. Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol. Cell. 2:397-403; Matsui, H., B. R. Grubb, R. Tarran, S. H. Randell, J. T. Gatzy, C. W. Davis, and R. C Boucher. 1998. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell. 95:1005-1015). Thus, if water were simply added to the airway surface, electrolyte concentrations would rapidly return to equilibrium values. However, if an osmolyte that has a low transepithelial permeability were added to the ASL, it might serve to lower the salt concentration. Somewhat analogous to this, the relatively impermeable osmolyte lactose allows the water-permeable mammary gland duct epithelium to maintain the lumenal NaCl concentration at 5-10 mM (Neville, M. C., P. Zhang, and J. C. Allen. 1995. Minerals, ions and trace elements in milk. In Handbook of Milk Composition. Academic Press. 577-675). Second, an osmolyte that is non-ionic would be required, because it is ionic strength which inhibits antimicrobial activity, not osmolarity (Travis, S. M., B. A. D. Conway, J. Zabner, J. J. Smith, N. N. Anderson, P. K. Singh, E. P. Greenberg, and M. J. Welsh. 1999. Activity of Abundant Antimicrobials of the Human Airway. Am. J. Respir. Cell Mol. Biol. 20:872-879; Neville, M. C., P. Zhang, and J. C. Allen. 1995. Minerals, ions and trace elements in milk. In Handbook of Milk Composition. Academic Press. 577-675). Third, the osmolyte should not provide a ready carbon source for bacterial growth. Fourth, the osmolyte should be safe in humans. Fifth, because many endogenous antimicrobials kill very quickly (Travis, S. M., B. A. D. Conway, J. Zabner, J. J. Smith, N. N. Anderson, P. K. Singh, E. P. Greenberg, and M. J. Welsh. 1999. Activity of Abundant Antimicrobials of the Human Airway. Am. J. Respir. Cell Mol. Biol. 20:872-879), even a transient decrease in ionic strength might be effective. Finally, a small reduction in the salt concentration, perhaps only 10 mM, might be beneficial because there is no unique relationship between antimicrobial activity and ionic strength; the lower the ionic strength, the greater the bacterial killing (Goldman, M. J., G. M. Anderson, E. D. Stolzenberg, U. P. Kari, M. Zasloff, and J. M. Wilson. 1997. Human xcex2-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell. 88:553-560; Bals, R., X. Wang, Z. Wu, T. Freeman, V. Bafna, M. Zasloff, and J. M. Wilson. 1998. Human xcex2-defensin-2 is a salt-sensitive peptide antibiotic expressed in human lung. J. Clin. Invest. 102; Travis, S. M., B. A. D. Conway, J. Zabner, J. J. Smith, N. N. Anderson, P. K. Singh, E. P. Greenberg, and M. J. Welsh. 1999. Activity of Abundant Antimicrobials of the Human Airway. Am. J. Respir. Cell Mol. Biol. 20:872-879; Singh, P. K., H. P. Jia, K. Wiles, J. Hesselberth, L. Liu, B. D. Conway, E. Valore, M. J. Welsh, T. Ganz, B. F. Tack, and P. B. J. McCray. 1998. Constitutive and inducible expression of xcex2-defensin antimicrobial peptides by human airway epithelia. Unpublished).
Current methods to treat CF infections are only partially effective and are not directed at the underlying defect. As can be seen from the foregoing, there is a need for a method of treating these infections which addresses the underlying defect. Furthermore, there is a general need for a method of preventing and/or treating epithelial infections.
An object of the invention is to provide a method for lowering ionic strength in body fluids.
Another object of the invention is to provide a method for killing infectious microbial cells by lowering the ionic strength of bodily fluids in which endogenous antimicrobials are found.
A further object of the invention is a method to lower ionic strength of body fluids by addition of a non-permeable, non-ionic osmolyte, such as xylitol.
An additional object of the invention is a method to prevent and/or treat epithelial infections.
These and other objects, features, and advantages will become apparent after review of the following description and claims of the invention which follow.
The present invention uses application of low permeability, non-ionic osmolyte(s) to allow endogenous antimicrobials to kill infectious microbial cells by decreasing ionic strength in fluids where the endogenous antimicrobials are found.
In CF, the low permeability osmolyte(s) are applied to the apical surface of CF airway epithelia to reduce the salt concentration in the surface liquid. The low permeability osmolytes allow CF epithelia to maintain and increase a transepithelial NaCl concentration gradient.
The preferred non-absorbable osmolyte is xylitol, a 5-carbon polyol. Xylitol has a low transepithelial permeability, is poorly metabolized by bacteria, and can lower the ASL salt concentration in both CF and non-CF airway epithelia in vitro.
When bacteria were deposited into xylitol-containing liquid covering CF epithelia, the bacteria were killed. In contrast, bacteria grew when they were deposited on untreated airway epithelia or epithelia treated with a saline solution. Xylitol is not an antibiotic on its own, instead it allows the killing of bacteria simply because it lowers the NaCl concentration of the surface liquid, thereby enhancing the activity of the endogenous antimicrobials.